MIIM30014 Lecture Notes - Sites of Infection PDF
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This document provides lecture notes on the sites of infection and implications associated with various portals of entry for viruses. It discusses topics such as respiratory, alimentary, and placental routes of viral infection, as well as the mechanisms of viral spread within the human body, including viremia and neural spread. The lecture notes also cover the importance of the immune response in determining the course of infection. The final section addresses the different types of viral transmission.
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Lecture 12 Sites of infection and implications associated with those portals Most infections have no consequence ○ Many particles never find a living cell to infect ○ Many are destroyed or inactivated as they enter the host ○ Man...
Lecture 12 Sites of infection and implications associated with those portals Most infections have no consequence ○ Many particles never find a living cell to infect ○ Many are destroyed or inactivated as they enter the host ○ Many infections never go further than one or two cells at the site of infection If infected, many infections are inapparent ○ No symptoms, but immune defences are activated ○ Viruses may be replicated and transmitted during these inapparent acute infections To cause infection of a host, virus must ○ Gain entry to body ○ Multiply and spread ○ Target appropriate organ To be maintained in nature, viruses must either be ○ Shed into the environment ○ Taken up by an arthropod vector or needle ○ Passed congenitally After initial acute infection, the course of infection is determined largely by the immune response of the host - clearance or persistence Tropism - anatomical localisation of infection that is initially determined by the receptor specificity of the virus The human body presents only a limited spectrum of entry sites for viral infection Most viruses enter via the epithelial cells of the mucosa because the epidermis of the skin is covered by dying cells covered with keratin - a hostile environment Respiratory tract ○ Most important site of entry ○ Acquired by aerosol inhalation or mechanical transmission of infected nasal secretions ○ Droplet size determines initial site of virus deposition More than 10μm - lodge in nose 81 5-10μm - airways Less than 5μm - alveoli of lower respiratory tract ○ Barriers to infection Mucus Cilia Alveolar macrophages Temperature gradient IgA ○ Viruses attach to specific receptors on epithelial cells ○ Can remain localised or spread further ○ Alimentary tract ○ Ingested viruses can either be swallowed or infect the oropharynx and then be carried elsewhere ○ Barriers to infection Sequestration in intestinal contents - constant movement of contents can allow contact with specific receptors Mucus Stomach acidity Intestinal alkalinity Proteolytic enzymes secreted by pancreas Lipolytic activity of bile 82 IgA Scavenging macrophages ○ Viruses that infect the intestinal tract are normally acid and bile resistant and do not have an envelope - coronaviruses are susceptible to these factors but are protected when ingested in milk or food and so can cause enteric infection ○ Some viruses cause diarrhoea, others do not cause disease in the intestinal tract but spread from there to cause systemic infection ○ If viruses do not have receptors for epithelial cells, need to enter via a breach in the epithelial surface - HIV, Hep B can infect via abrasions of the rectal route ○ Entry via GI tract may involve Local infection - rotavirus, coronavirus, adenovirus, norovirus Invasion of the host to produce systemic illness - enteroviruses, hep A Due to invasion of tissues underlying the mucosal layer Transcytosis through M cells ○ Virus survival depends on Acid stability Resistant to bile salts Inactivation by proteolytic enzymes - in some cases this is a requirement ○ Most are non-enveloped (naked viruses generally more stable) ○ 83 Placenta ○ Several viruses including Zika virus, rubella virus, cytomegalovirus, and HSV can cross from mother to foetus ○ Dependent on multiple variables including virus type and foetal age ○ Syncytiotrophoblasts are crucial cells that lie at the maternal-foetal interface and are resistance to infection by many viruses ○ Routes of vertical transmission across the placenta could include breaks in the syncytial cell layer, targeting other cell types such as cytotrophoblasts or extravillous trophoblasts or bypassing this by mechanisms such as direct antibody-mediated transcytosis ○ Local vs systemic spread Local - confined to organ of entry Systemic - involves many organs Many viruses multiply in epithelial cells at site of entry, produce a spreading infection, then shed directly to exterior ○ Respiratory - influenza, rhinoviruses and RSV ○ Gastrointestinal - rotaviruses and noroviruses ○ Dermatologic - papillomaviruses Surface infection - failure to spread to deeper tissues - e.g. influenza virus in respiratory epithelium Infection through the apical side, exit by the apical side and so remain localised In systemic, polarised infection of epithelial cells and spread 84 ○ Targeting a viral budding to apical or basal surfaces of polarised cells may define subsequent spread - could be by viral glycoproteins - secreted proteins carry signals for targeting Invasion of deeper tissues - e.g. HSV Mumps virus - systemic ○ Primary viral replication in epithelial cells of upper respiratory tract ○ Receptor is sialic acid ○ Systemic infection involving mostly all organs including CNS ○ Mild meningitis common, encephalitis uncommon ○ Characteristic salivary gland involvement Measles virus - systemic ○ Primary viral replication in epithelial cells of upper respiratory tract ○ Receptors include CD150 and CD46 ○ Infects local macrophages, lymphocytes and DC cells, then in draining LN ○ Enters circulation and amplified in lymphoid tissue ○ Returns to epithelial cells in lung and mouth ○ Highly contagious ○ Transient immunosuppression Spread of infection within the body Haematogenous spread ○ Tissue invasion from the blood ○ Barrier to entering tissues - capillary endothelial cells 85 ○ Some viruses can replicate in endothelial cells and progeny virus is released into tissues ○ Some viruses are transported across these cells by transcytosis ○ In some tissues these are not joined by tight junctions and virus can pass between them - e.g. mumps in choroid plexus of brain ○ Some viruses cross to tissues in monocytes and lymphocytes as part of normal cell trafficking Neural spread ○ Via peripheral nerves - HSV, rabies, varicella ○ Uncoated nucleocapsid is carried passively along axons or dendrites ○ Multiplication in body of nerve and released progeny can cross synaptic junction - eventual entry into the CNS ○ Viruses protected from attack by CTL as nerve cells do not have class I molecules ○ Other viruses enter CNS directly via the bloodstream as for other tissues ○ Rabies virus Rabies virus replicates at the site of infection until enough viral particles come in contact with sensory or motor nerve cells Viruses enter the axons where they are not myelinated and move up towards the neuron’s nucleus by retrograde axoplasmic flow Rabies virus capitalises on this mechanism to reach the spinal cord Transport along peripheral nerves can occur in both a forward (anterograde) or backward (retrograde) direction This can be from the site of infection to other sites within the body 86 Or from reservoir sites back to epithelium (Herpesviruses) ○ Herpes virus Varicella-zoster virus (chickenpox in children) can utilise both viremia and neural spread at different stages Vertical (host to progeny) ○ Viruses may also infect the foetus ○ Must cross the placenta ○ Death and abortion can occur by cytocidal viruses like smallpox ○ Developmental abnormalities can occur by non-cytocidal viruses like rubella and CMV ○ Viruses may also infect the baby at birth HSV, varicella and CMV - infected birth canal Coxsackie B - faecal contamination Viremia can lead to immunological tolerance (HBV) and carriage state Viremia - understanding and different types The presence of infectious virus particles in the blood - virions may be free in the blood or contained within infected cells such as lymphocytes Types ○ Active viremia - produced by replication The presence of newly synthesised virions in the blood Free particles or in infected lymphocytes ○ Passive viremia - viral particles are introduced into the blood without viral replication at the site of entry Viruses may be free in plasma 87 ○ Primary and secondary phases ○ Produced by infected vascular endothelium or released in large amounts from e.g. liver and spleen ○ Neutralised by developing Ab response and removed by macrophages - usually 1-2 weeks Viruses could be cell associated ○ E.g. measles and dengue spread by monocytes, HIV spreads in CD4+ T cells ○ Can persist from months to years if viral genome becomes latent to avoid CTL attack Virus particles move from the blood into tissues at different stages of the infection ○ This offers a window of opportunity for our immune response when in blood, particularly antibody ○ But the virus avoids antibody detection when in the tissue - cell mediated immunity is key here ○ Pathogenesis of poxvirus (right)^ Transmission of spread Respiratory tract 88 ○ Either aerosols generated by sneezing or coughing or nasopharyngeal secretions spread from mouth to hand to mouth ○ Viruses causing acute respiratory diseases (e.g. influenza virus and rhinovirus) are spread at high titre but for short periods - less than a week ○ Measles - peak of infectiousness is prior to rash Oropharynx and gastrointestinal tract ○ Enteroviruses can be shed from pharyngeal fluids as well as faeces - e.g. polio after replication in lymphoid tissue of tonsils and Peyer’s patches ○ Virus can be excreted into faeces from epithelial cells of the intestinal tract - e.g. rotavirus the liver via the bile duct - e.g. HepA virus Skin ○ HPV: warts virus exit only from top layers of skin (not internally) and transmitted by mechanical contact ○ VZV: shed from virus-filled vesicles (virus can become aerosolized and transmitted via the respiratory route) Blood ○ Usually viruses that produce persistent viremia eg. HBV, HCV and HIV ○ Cytomegalovirus (CMV) in acute or reactivated state (transfusions) ○ The flaviviruses Dengue and West Nile virus where virus levels in the blood are sufficient for man to mosquito to man transmission ○ Most arboviruses are transmitted by insect vectors from bird to bird or among small mammals ○ If the vector should feed on a human the virus may be transmitted (epizootic infection) with resulting illness ○ Direct transmission from human to human does not occur and vector transmission among humans usually does not occur because levels of virus in the blood are very low or undetectable with the above exceptions Urine, milk, oral and genital secretions ○ CMV: urine, saliva, tears, breast milk, semen, and cervical secretions (infection is usually inapparent therefore dangerous for pregnant mother -> foetal abnormality) 89 ○ ZIKV: arbovirus but recently shown to be present in urine and transmitted in semen ○ HIV, HTLV-1 - milk ○ HPV, HSV-2 - semen, and cervical secretions ○ Mumps virus, rubella virus - milk ○ EBV, rabies virus - saliva ○ HIV, HBV - semen (HIV free or in infected cells - average ejaculate of infected male has a million lymphocytes of which 100-10,000 may carry the viral genome) 90 Lecture 13 Understand and describe the determinants of tropism Availability of receptors for the virus ○ Key factor determining initial susceptibility of a cell type to a particular virus ○ But many viruses have receptors that are ubiquitous and yet have restricted tropism ○ ICAM-1 is expressed more at higher altitudes and down regulating with alcohol ○ Need to also consider permissivity (cell has all the right intracellular gene products to replicate viral genome), accessibility (can virus access susceptible cells) ○ Consider whether local factors prevent or allow infection of a tissue Local factors ○ Optimal temperature for replication - e.g. rhinovirus restricted to URT ○ Stability in extremes of pH - e.g. low pH of stomach, high pH of intestines ○ Ability to survive destruction of lipids by bile and proteases produced by pancreas In some cases, exposure to these factors is essential for infection (e.g. Rhinovirus vs poliovirus - poliovirus is activated by low pH but rhinovirus is destroyed, yet both are enteroviruses) Factors affecting accessibility include ○ Ability to replicate in macrophages and lymphocytes or travel free in blood - gain access to many tissues ○ Polarised release (apical vs basolateral) - hard to gain access to deeper tissues if only released from apical side of cell Availability of cleavage-activating proteases 91 ○ Influenza HA is made as a single polypeptide chain which must be cleaved next to the fusion peptide for the virus to be infectious ○ At pH 5 in the endosome, the fusion peptide must be free to change confirmation ○ Cleavage is by tryptase (previously called tryptase clara) which is only secreted in the respiratory tract ○ Some highly virulent avian influenza virus strains contain insertion of multiple basic amino acids at HA cleavage site - a recognition site for furin ○ Permits cleavage by ubiquitously expressed intracellular proteases (furins- Golgi) ○ Infectious viruses are released from cells and can infect many organs ○ Avian influenza viruses (H5N1) isolated from 16 people in Hong Kong contained similar amino acid substitutions at the HA cleavage site - highly pathogenic avian influenza viruses (HPAI) ○ For SARS-CoV entry into a host cell, its S protein needs to be cleaved by cellular proteases at 2 sites, termed S protein priming ○ Externally, S protein priming is performed by the serine protease TMPRSS2 - internally, performed by Cathepsin L in the endosome ○ ACE2 and TMPRSS2 are only expressed together in the respiratory tract, gut enterocytes of ileum and colon and liver ○ This restriction is somewhat overcome by the furin-active site with spike of SARS-CoV-2 that may occur during exit or entry Genome transcription requirements ○ E.g. papillomavirus - the skin is the only tissue that can support the complex cascade of transcription factors to support the complete replication cycle of the virus ○ Virus commences genome replication in the germinal cells in the basal layer of the skin BUT these cells produce proteins that block transcription of late structural genes ○ Capsid proteins can only be made once the germinal cells have differentiated and moved outwards to become permissive for viral gene transcription and translation 92 ○ tRNA availability defines whether some proteins can be translated or not - papillomavirus uses these rare tRNAs and since structural proteins are not produced until the skin cells travel further up and are ready to slough off, the immune system does not recognise the presence of the virus Define the effects of virus infection on the cells they infect Cytocidal virus ○ Shutdown of cellular protein synthesis Most cytocidal viruses code for proteins for this purpose (e.g. adenovirus protein inhibits transport of cellular mRNA from nucleus to cytoplasm; poliovirus protease 2A cleaves eIF-4G so cellular mRNA cannot be translated) Others by competition of viral and cellular mRNA for ribosomes ○ Shutdown of cellular nucleic acid synthesis A consequence of host protein shutdown or more specific mechanisms E.g. poxvirus encodes a DNase Cytopathic effects of viral proteins ○ Accumulated viral proteins can be cytotoxic ○ Cytoplasmic inclusion induced by virus infection as viewed by electron microscopy Formation of synctia ○ Viral proteins inserted into plasma membrane may Cause cell fusion Leads to changes in membrane permeability and cell lysis Apoptosis ○ A series of events leading to death that is carried out by cells when their normal cell function is compromised ○ Host escape mechanism leads to cell death and inflammation by cysteine proteases (caspases) Induction of cytokines Infected cells release proteins that are subsequently presented by MHC II 93 Activation of CTLs Cells may lose their ability to perform particular functions ○ One of the best ways to disable the immune response is to destroy the bodies cells that mediate the immune response ○ HIV is one of the best at this as it can infect both macrophages and T cells Non-cytocidal virus ○ Cells may lose their ability to perform particular functions ○ E.g. LCMV causes a non-lytic persistent infection of growth hormone producing cells in the pituitary gland and transcription of growth hormone mRNA is specifically reduced ○ E.g. rhinoviruses and cilial stasis in respiratory epithelium ○ E.g. SARS-CoV-2, ACE2, inflammation and the immune response SARS-CoV-2 appears to impact at many levels Can be direct effects on cells - SARS shuts off host translation Dysregulation of the RAAS pathway Dysregulated inflammation Dysregulated immune response - timing of the innate immune response is very important Targets for immune attack ○ MHC I and CD8+ T cells ○ Normally CD8+ can immediately recognise infected cellsand destroy them via recognition of antigenic peptides within the MHC I or via release of enzymes into the infected cells Transformation ○ Some viruses encode oncogenes whose expression in the virus infected-cell is associated with tumour production ○ These gene products generally alter the regulation of the cell cycle - uncontrolled cell proliferation, reduced contact inhibition - cells pile on top of each other Understand the different mechanisms of disease progression Viral induced damage to tissues and organs ○ Death as a direct result of viral replication 94 ○ Loss of function ○ Disease may result directly from the cell damage resulting from viral replication - cytocidal virus E.g. tissue specific cell killing in rotavirus diarrhoea (enterocytes), and paralytic polio virus infection (neurons in spinal cord) ○ Most non-enveloped viruses rely on the cell lysing for their release but sometimes the replication cycle of enveloped viruses also leads to death Host resistance ○ Genetic factors Inherited defects e.g. absence of Ig class Polymorphisms in genes controlling immune responses e.g. MHC genes Interferon-inducible genes (MxA and MxB; resistance to influenza and other negative strand RNA viruses and HIV) Receptor genes e.g. CCR5 ○ Non-genetic factors Age - newborns and the aged are more susceptible to severe disease (immature and waning immune response) but young suffer less from immunopathology; elderly also succumb more easily due to waning immunity - e.g. West Nile virus infection Malnutrition - decreases resistance; e.g. Vitamin A deficiency increase susceptibility to, and severity of, measles infection Hormones, pregnancy - males and pregnant women more susceptible → androgens in males and oestrogens in females, modulate several aspects of host immunity Dual infections - may result in more severe disease; e.g. HIV and HHV-8 (Kaposi sarcoma) Consequences of the immune response ○ Immunopathology Most viral infections elicit a powerful immune response with infiltration of lymphocytes and macrophages, release of cytokines and inflammation 95 Symptoms such as fever and fatigue (IL-1, IL-6, TNF acting on hypothalamus), muscle pain, nausea and enlargement of lymph nodes all have an immunological basis Elevation of temperature is a powerful weapon as most microbes cannot tolerate high temperatures - enzymes and proteins misfold and thus don’t operate effectively ○ Antibody-mediated pathology Antibody dependent enhancement of infection by FcR-mediated uptake of virus-antibody complexes into macrophages and monocytes E.g. Dengue haemorrhagic fever/shock syndrome Antigen-antibody complexes deposited in the kidney can cause glomerulonephritis and in the blood vessel, vasculitis E.g. Hep B in chronic carriers ○ T cell mediated pathology CD4 T cell mediated responses Responsible for some viral rashes e.g. measles - type IV delayed type hypersensitivity in which T cells mediate an immune response to the virus, involving inflammation, in the endothelial cells of the small blood vessels in the skin Induce cytokines that recruit eosinophils - responsible for bronchiolitis in infants with respiratory syncytial virus (RSV) infection CD8 T cell mediated responses 96 Contributes to liver damage in Hep B virus infection ○ Lysis of hepatocytes, recruitment of monocytes and neutrophils ○ Yellow skin and eyes are a sign of liver damage Old RBCs are destroyed by macrophages in spleen Heme from haemoglobin is converted to bilirubin - yellow Normal conditions → bloodstream → liver for secretion in bile → faeces Damaged liver cells cannot carry out these processes and so bilirubin is in tissues and none in faeces ○ Immunosuppression E.g. HIV replicates in CD4 T cells and kills them and in monocytes and inhibits their function E.g. Norovirus replicates in macrophages and dendritic cells affecting their function during immune communication E.g. Measles - temporary immunosuppression from nonproductive replication in T cells and macrophages; suppression of proliferation of non-infected T cells by engagement of measles surface glycoproteins with cell surface receptors (CD46) that result in suppression of IL-12 Leads to susceptibility to secondary infections ○ Autoimmunity Outcomes of virus infection ○ Fatal - viral diseases where man is not the natural host have very high mortality rates, e.g. Ebola ○ Full recovery - virus is completely cleared by host’s immune system, e.g. influenza ○ Recovery but permanent damage - virus cleared but left with symptoms, e.g. poliomyelitis, cancer ○ Persistent infection - virus not cleared and can resurface to cause disease, e.g.Herpesviridae(cold sores, shingles) 97 Lecture 14 How does viral virulence contribute to pathogenesis How can the events of replication directly affect pathogenic outcomes Roles of viral proteins that not only influence replicative abilities but also pathogenic outcomes Viral virulence - capacity of a virus to cause disease in a host Virulence can be quantified ○ Mean time to death in controlled conditions not human population ○ Mean time to appearance of symptoms ○ Measurement of fever, weight loss - there is a limit, can’t go down a certain point for weight loss and instead need to cull them ○ Measurement of pathological lesions (poliovirus); reduction in blood CD4+ lymphocytes (HIV-1) ○ Measurement of survival Mice inoculated intracerebrally with type 1 or type 2 poliovirus Poliovirus type 1 cannot infect the mouse CNS, only type 2 is neurovirulent Genetic determinants of virulence ○ A major goal of animal virology is to identify viral and host genes that control virulence ○ To identify viral virulence genes, it is necessary to compare similar viruses that differ only in their degree of virulence 98 ○ Viral virulence genes can be placed in one of four general classes Gene products that alter the ability of the virus to replicate Genes that encode proteins affecting both viral replication and virulence can be placed in one of two subclasses ○ Viral mutants that exhibit reduced or no replication in animal and in many cultured cell types - reduced virulence results from failure to produce sufficient numbers of virus particles to cause disease ○ Mutants that exhibit impaired virulence in animals, but not replication defects in cells in culture - such host range mutants provide valuable insight into the basis of viral virulence, because they identify genes specifically required for disease - can grow well in lab but the moment it is introduced to animal, there is no virulence Gene products that modify the host’s defence mechanisms Viral proteins that sabotage the body’s intrinsic defences and innate and adaptive systems ○ Some of these viral proteins are called virokenes - secreted proteins that mimic cytokines (will bind to receptors but will not initiate the signalling cascade → dysfunctional), growth factors, etc OR viroceptors - homologs of host receptors 99 Mutation in genes encoding either class of protein affect virulence - but these genes are not required for growth in cell culture Most virokines and viroceptors have been discovered in the genomes of large DNA viruses - RNA viruses are restricted in their coding capacity Genes that enable the virus to spread in the host Mutation of some viral genes disrupt spread from peripheral sites of inoculation to the organ where disease occurs ○ After intramuscular inoculation in mice, reovirus type 1 spreads to the CNS through blood, while type 3 spreads by neural routes Studies of viral recombinants between type 1 and 3 indicate that the gene encoding the viral outer capsid protein δ1, which recognises the cell receptor and determines route of spread Toxic viral proteins - they’re not toxins like in bacteria; but some of the viral proteins are toxic to cells Some viral gene products cause cell injury directly and alterations in these genes reduce viral virulence Evidence of their intrinsic activity is usually obtained by adding purified proteins to cultured cells or by the synthesis of proteins from plasmids or viral vectors ○ NSP4 is a non-structural glycoprotein that participates in the formation of a transient rotavirus envelope as the particles bud into the endoplasmic reticulum ○ When this protein is produced in insect cells, it causes an increase in intracellular calcium concentration 100 ○ When fed to young mice, NSP4 causes diarrhoea by potentiating chloride secretion ○ NSP4 acts as a viral enterotoxin and triggers a signal transduction pathway in the intestinal mucosa ○ Effect of noncoding sequence on virulence Attenuated Sabin vaccine strains of poliovirus contain a mutation in the 5’ noncoding region (NCR) that reduces neurovirulence Deletions within the long poly(C) tract in the 5’ NCR of mengovirus reduce virulence in mice It’s not just gene products that impact virulence and replication, it is also the noncoding sequence and secondary RNA structures RNA viruses ○ The masters of error-prone replication - RNA pol cannot correct errors ○ Average error frequency - 1 in 104 or 105 nucleotidespolymerised ○ In a 10 kb RNA virus genome, a mutation frequency of 1 in 104 results in about 1 mutation per genome ○ Most errors are deleterious → mutations in essential regions ○ Others can be beneficial - mutations on structural proteins that slightly affect conformation but not function Antigenic drift ○ In this situation, the viral RdRp is able to switch template if two different viral strains have infected the same cell - very rare but does happen ○ This creates a chimeric viral genome that contains sequences from both strains ○ Generally occurs when subgenomic RNAs are produced - a lot of template that the RdRp can use 101 ○ This process is inherently different to reassortment, which is the packaging of mixed RNA segments during the assembly of segmented RNA viruses ○ SARS-CoV and IFN ○ Early IFN signalling is essential to reduce viral titre and ensure a mild progression ○ Delayed IFN signalling leads to increased viral titres and a severe disease ○ If there is no IFN signalling, the viral titres are well controlled and the disease outcome is still better ○ Dysregulated type I interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in SARS-CoV infected mice ○ Severe disease Delayed innate response/IFN-1 Inflammatory monocyte-macrophage response (IMM) ○ Mild symptomatic infection Early or suppressed innate/IFN-1 ○ Many SARS-CoV 2 proteins modulate innate immunity Block multiple arms of type I interferon response to dampen the production of IFN-beta, including non-structural proteins (nsp) and accessory gene proteins (ORF) Activation of the interferon stimulated genes that are important for antiviral activities are blocked at multiple levels ○ SARS-CoV 2 suppresses IFN-beta production mediated by NSP1,5,6,15, ORF6 and ORF7b but does not suppress the effects of added interferon ○ SARS-CoV 2 shuts down host cell translation preventing cytokine production ○ Activation of IFN signalling is impaired ○ This is in part attributable to the viral protein NSP1 - the very first gene product that is synthesised and then ensures viral translation dominates 102 ○ STAT1 is a transcription factor that needs to be phosphorylated to translocate to the nucleus and initiate the innate immune response - it is found that in the presence of IFNα and SARS-CoV infection, not only is STAT1 production limited, but the phosphorylation is also decreased which then impacts the extent to which innate immunity is established against the infection Pathogenesis of Filovirus infection ○ Ebola and Marburg virus ○ Haemorrhagic fever, shock and death ○ Hypothesis - shock is often associated with release of cytokines by macrophage/monocyte ○ Ebola virus Most lethal virus known to humans - kills 50-90% of the infected individuals Outbreaks occurred all over central Africa Believed to be primate deadly, Monkeys have infected many doctors, scientists, and monkey care handlers ○ Ebola virus - recent history Genetic analysis indicates that it is closely related to variants of Ebola virus (species Zaire Ebolavirus) identified earlier in Congo and Gabon Zaire Ebolavirus normally kills 80% of infected individuals ○ Ebola virus - symptoms Abrupt and unexpected Incubation between 2-21 days - signs of disease Starts with red eyes, leads to fever, headache, flu-like symptoms, fatigue, internal/external bleeding, massive haemorrhage 103 The infected body is also corroding away from the inside ○ Ebola virus - transmission Ebola is transmitted through bodily fluids and/or direct contact with infected individuals Believed to spread to human populations through contact with infected primates The suspected natural sources of the virus are certain species of fruit bats - they have been found to carry the virus, but they themselves are asymptomatic Outbreaks of Ebola virus are often traced to an individual that has handled a gorilla or chimpanzee carcass It is common for the virus to then spread to family members or hospital workers because of their close proximity to the victim The virus spreads to people that come into contact with these patients’ blood or contaminated medical equipment Because Ebola kills its victims so quickly and the outbreaks usually occur in isolated areas, the disease does not typically spread very far Also, infected persons do not have time to carry the disease very far ○ Ebola virus - cures/treatment No cure Due to extreme biohazard, doctors must wear level 4 containment suits Some towns put the diseased in a straw hut and then burn it down when they’re dead - simple yet effective If contained properly, ebola is not that dangerous ○ Ebola virus - pathogenesis The Ebola gene products have obvious roles in the virus life cycle Structural and non-structural The Ebola glycoprotein (GP) is a major structural component and an antibody target The VP35 protein is an essential polymerase cofactor The VP24 protein plays a role in virus assembly and budding 104 However all these proteins also impart additional functions in host cells An additional Ebola GP (glycoprotein) species is generated via transcriptional editing via addition of an extra adenosine residue altering the reading frame and thus producing a larger protein species results in a GP species that now contains a membrane spanning domain and is incorporated into virions Both of these proteins look structurally similar at their N-termini The sGP (short GP, 75%) serves as a decoy and “mops-up” or sequesters circulating antibody to avoid neutralisation Additionally, the virion-associated GP requires furin cleavage for activation The VP35 and VP24 additionally disarm immune activation in Ebola virus-infected cells VP35 prevents activation of IRF3 and thereby restricts the transcription of and production of IFN - directly involved in signalling cascade VP24 prevents the nuclear translocation of the transcription factor STAT1, to impede the transcription and production of interferon stimulated genes (ISGs) of which most are antiviral A multifaceted approach to promote virus survival in cells by incorporating multiple functions into your gene products – maximises the restricted coding capacity of RNA viruses 105 TNF-α can induce necrosis (via apoptosis) and is a potent pro-inflammatory cytokine - when too much is produced a detrimental rather than beneficial state is induced In this case destruction and vascular instability result from TNF-α over production 106 Lecture 15 Describe the key molecules responsible for virus detection in cells and their specificity - TLRs, RLRs, DNA sensors Ligand binds to receptor → adaptor molecule activated → cytosolic transcription factor activated → translocates to nucleus → induces gene transcription Activation of specific molecules occur via oligomerisation and/or phosphorylation Toll-like receptors (TLRs) recognise certain pathogen associated molecular patterns (PAMPs) ○ TLR3 - double stranded RNA ○ TLR7 - single stranded RNA ○ TLR8 - single stranded RNA ○ TLR9 - Herpesvirus infection ○ Ligand induced dimerisation of TLR leads to induced assembly with adaptor signalling molecules ○ MyD88 pathway and TRIF pathway activate transcription factors and MAP kinases ○ Both lead to the activation of NFκB and IRF6 ○ TLR3 also activates IRF3 107 RIG-I (retinoic acid inducible gene I) and MDA5 (melanoma differentiation associated gene 5) are cytosolic RNA helicases ○ Function is to recognise viral RNA species ○ Specificity exists in what they recognise ○ Initiates a downstream signalling pathway ○ The interaction of viral RNA with RIG-I or MDA5 induces dimerisation and interaction with the adaptor protein MAVS that is located on the mitochondrial membrane ○ The interaction is facilitated via CARD (caspase activation and recruitment domain) domains and stimulates the activation of transcription factors IRF3/7 and NFκB Cytosolic DNA is foreign to human cells and therefore acts as a danger signal for virus infection DNA receptors in the cytoplasm monitor and detect foreign DNA and activation of these receptors result in the production of cytokine transcription or triggering the inflammasome which leads to cell death cGAS (cyclic GMP-AMP synthase) is a host protein that recognises foreign DNA in the cytoplasm ○ Generates cGAMP (cyclic guanosine monophosphate adenosine monophosphate) that binds to STING in the ER ○ This activates TBK1, IRF3 and NFκB to produce IFN and cytokines ○ Evidence also suggests that cGAMP is released from cells and can induce an antiviral state in neighbouring cells Identify the key adaptor molecules - MAVS, MyD88, TRIF, cGAS-STING Discussed above Identify the key transcription factors and their function - IRF3/7, NFκB, STAT1/2 Both NFκB and IRF3 are transcription factors and must enter the nucleus to induce their effects IRF3 leads to transcription of type I interferons - IFNα/β NFκB results in the transcription of pro-inflammatory cytokines - IL-6 and TNFα 108 Define the production of ISGs and their roles as antiviral proteins Cytokines are low molecular weight proteins with important regulatory functions in the innate and adaptive immune system Upon secretion, they bind to specific receptors on the producer cell or other cells Subsequent intracellular signalling cascades alter gene expression in target cells Interferons initially function locally as antiviral defence In larger quantities, interferons enter circulation and have global effects - sleepiness, lethargy, muscle pain, no appetite, nausea Interferon induced Jak-Stat pathway ○ 109 Interferon stimulated genes (ISGs) are proteins that are only made upon IFN stimulation - only produced during virus infection There are 200-300 ISGs The MxA protein is an example of ISG and is only found in the cytoplasm ○ Specific only to virus infection ○ Recognises viral capsids ○ Sequesters nucleocapsid in bundles ○ Extremely potent against influenza virus ○ Widely used as marker of infection OAS and RNaseL are other examples of ISG that specifically detect viral RNA species ○ Induces the OAS monomer to form a tetramer ○ The OAS tetramer makes 2’-5’-adenylic acid (a nucleotide) ○ Induces the inactive RNaseL monomer to dimerise and become active ○ Active RNaseL can then degrade the viral RNA ○ RNaseL is always present in cells but in very small amounts and in inactive state ○ Both OAS and RNaseL production is induced by IFN Protein kinase R (PKR) is another ISG that specifically detects viral RNA species ○ The inactive PKR monomer contains a dsRNA binding domain ○ Upon binding to dsRNA, PKR is phosphorylated and dimerises to become active ○ Active PKR then phosphorylates the host cell translation factor EIF2α and inhibits it - stopping all host cell translation and consequently, virus protein translation ○ The PKR mechanism is a shut-down mechanism to halt virus replication and has global effects Understand how viruses counteract the activation and function of the innate immune system 110 Interfere with RNA recognition, helicase activation, MAVS signalling or prevent activation of transcription factors → inhibition of IFN production Inhibition of IFN signalling ○ West Nile virus prevents phosphorylation ○ SARS CoV prevents nuclear import ○ Measles and mumps and most likely noroviruses prevent nuclear import ○ West Nile virus also redistributes cholesterol from the plasma membrane which disables signalling via the IFN receptor Can be rescued via exogenous addition of cholesterol Inhibition of PKR ○ Almost every virus has devised a way to prevent PKR activation and activity ○ EBV, HIV, adenovirus - fake RNAs ○ Poliovirus - cleavage of inactive PKR monomer ○ Poxviruses and reoviruses - prevent dimerisation and/or phosphorylation ○ HCV, HSV, poxvirus - phosphorylates 111 Lecture 16 Mammalian cells produce a variety of cellular RNAs from nuclear DNA template ○ Spliced mRNA that codes for protein ○ Non-coding RNAs Directing protein translation Regulating gene expression Catalyse mRNA splicing Facilitate RNA base modification ○ RNA is readily degraded by RNase and pH 112 RNA modifications modulate expression and innate responses ○ ○ Alternative mRNA splicing is important for generating diversity Innate recognition of pathogen nucleic acids ○ PAMPS recognised by pattern associated recognition receptors ○ Includes DNA, RNA and nucleic acid modifications ○ Activate cellular signalling pathways ○ Resulting in production of interferon and cytokine production ○ Non-specific and generic mediators Eukaryotes are when RNA viruses start emerging DNA viruses in bacteria and archaea are dealt with CRISPR, while in lower order species like plants, RNA interference is observed; interferons are observed in mammalian vertebrates CRISPR-Cas9 edits eukaryotic DNA RNAi (RNA interference) became important for plants and fungi 113 Short-dsRNAs drive an important pathway for "genetic immunity" in plants and invertebrates ○ Common to both types of silencing is the production of ~21 nucleotide siRNA which contains a perfectly duplexed 19 base-pair (bp) double helix with 2-nucleotide single-stranded tails at the 3’ ends. Often has uridine overhangs at the end ○ These are called short interfering RNAs, or siRNAs, because they interfere with the expression of genes or RNA transcripts which have complementary sequences. ○ In plants and invertebrates, the long dsRNA (e.g., viral replicative intermediate RNA) is processed by an enzyme → Dicer recognises the dsRNA and gets processed into siRNA ○ siRNA loads into RNA-induced silencing complex and finds the original viral RNA and binds to it and causes degradation ○ The siRNA then is recycled RNAi results in enzymatic cleavage while microRNAs results in stoichiometric block In higher species of animals, the siRNA is known as microRNA Similar dsRNA based gene silencing later observed in invertebrate animals 114 Human cells naturally control their own gene expression using miRNA ○ miRNA are ~23 nt long imperfectly duplexed RNAs ○ Expressed by all metazoan eukaryotes ○ Some miRNA are conserved across phyla ○ Natural target sequence prediction is difficult due to G-U wobble pairing in RNA ○ Initially encoded as pre-mRNA - 80 nt RNA stem loop, sometimes in clusters ○ 40% miRNA are encoded in introns, despite only 2% protein coding DNA ○ miRNA repress mRNA translation and also other activities Both the IFN-I & miRNA systems operate in vertebrates ○ In mammals, the long imperfectly duplexed RNA is processed by an enzyme → Dicer ○ One strand becomes the “guide strand” and is assembled into the RNA-induced silencing complex, or “RISC” Sequence and structure selection Strand with the less-tightly base paired 5’ end is incorporated in RISC and becomes the “guide strand” ○ Argonaut proteins in RISC inhibit gene expression ○ Ago2 is an RNA endonuclease (Slicer) ○ Ago1 is an RNA transcription inhibitor (Silencer) ○ 115 ○ Transcriptional suppression nor translation ○ Know about argonaut proteins (Ago1/2), Drosha, TRBP, Dicer ○ GW182 is responsible for loading the RNA to the RISC In mammals, interference of gene expression without innate IFN-I responses occurs only by short dsRNA ○ In plants and invertebrates, long dsRNA is the main substrate for the production of small effector RNAs ○ In mammals, RNA silencing pathways are only triggered by short RNAs of 23 nt Short interfering RNAs - siRNA (produced artificially for therapy) Short hairpin RNAs - shRNA (also produced artificially by viral vectors) Micro RNAs - miRNA (produced in nature) Others - repeat-associated small interfering RNAs (rasi RNAs); Piwi-interacting RNA (piRNA) Mechanisms for miRNA suppression of gene expression ○ miRNA repress protein translation at initiation, elongation, termination and release ○ miRNA induce degradation of new forming peptide ○ mRNA degradation only occurs when a high level of duplex is formed, so usually by siRNA not miRNA ○ Transcriptional silencing - several mechanisms 116 Chordates (i.e. humans) have a protein mediated response pathway to virus derived RNA PAMPs leading to expression of type I IFNs and ISGs Nematodes, arthropods and plants use RNA PAMPs directly to generate virus-derived interfering RNAs (viRNAs) which can be loaded into an RISC to mediate targeted silencing and cleavage of viral RNA Nucleic acid-mediated activities in viral replication/host defence ○ Long dsRNA activates interferon-inducible proteins PKR and 2-5’ OAS leading to translation arrest and eventually cell apoptosis Adenoviral VA1 RNA is a decoy/fake RNA with diverse effects on innate PKR-mediated and RNAi pathways ○ Blocks nuclear export of pre-miRNAs ○ Inhibition of Dicer ○ Inhibition of PKR activation Potential mechanisms where miRNAs affect viral replication ○ Viral miRNA inhibits viral mRNA expression ○ Viral miRNA inhibits host cell mRNA expression ○ Host cell miRNA inhibits viral mRNA expression ○ 117 DNA viruses that encode miRNAs ○ Herpesviridae- hCMV, KSHV, EBV, MHV68, rLCV ○ Adenoviridae- hAV ○ Polyomaviridae- SV40 Control of gene expression by cell and viral miRNA - HSV ○ HSV-1 latency activated transcript (LAT) encodes a miRNA that down-regulates cellular TGFβ and SMAD3 that now makes infected cells relatively resistant to CTL killing by rendering of cell programmed death pathways defective ○ These transcripts don’t code for any protein since it is latent ○ TGFβ and SMAD3 are important in initiating apoptosis SV40 miRNA target T proteins to promote the switch from early to late gene expression and reduce apoptosis and CTL killing ○ ○ Acts similar to siRNA since it is perfectly duplexed and complementary ○ A facilitator of early to late switch - a post transcriptional selection decay Cellular microRNAs contribute to HIV-1 latency in resting primary CD4+ T lymphocytes ○ Important to switch from productive active infection to latency Cellular miRNA silence expression of viral RNA - Primate Foamy virus-1 ○ Human miR-32 inhibits PFV-1 replication ○ The viral Tas protein inhibits the production of cellular RNAi ○ 118 Liver specific cellular miR-122 binds the 5’ UTR of HCV RNA, stabilising it and enhancing expression ○ miRNA can be generated from RNA virus mRNAs Cellular restriction factors for HIV ○ Intrinsic antiviral proteins such as Tetherin, Trim5a, SAMHD1 and APOBEC3 exist that restrict lentiviral replication and inhibit infection by various mechanisms ○ Tetherin is an ISG and anchors to the cell membrane and prevents viral particles from entering the cell ○ The HIV protein Vpu inhibits tetherin ○ The cytidine deaminase APOBEC3 induces lethal G → A hyper-mutations of the viral genome while in ssDNA - identifies viral RNA, Gag and Pol proteins Error catastrophe - the right protein structures will not be coded for ○ The HIV Vif protein degrades APOBEC3 by the proteasome degradation pathway ○ ○ 119 Lecture 17 To understand the components and their roles during the adaptive immune response - T cells, NK cells, Abs, TCR, MHC class I, cytokines, inherent cell responses Neutralising antibodies ○ Biggest defence against virus infection ○ Binding can be to epitopes that facilitate entry i.e. receptor binding domain (RBD) can restrict endocytosis or uncoating ○ Can physically induce aggregation to facilitate immune clearance ○ Fc mediated uptake into mononuclear cells generally works ADE re DENV - antibody dependent enhancement (severe outcome) ○ 120 TNF- is not only a proinflammatory cytokine but can also induce antiviral effects when it binds to its receptor on infected cells; within seconds the combination of TNF binding and viral infection triggers the process of apoptosis (programmed cell death) Apoptosis is cell death mediated by cysteine proteases (Caspases) ○ The dying cells release apoptotic bodies containing viral proteins that are taken up by APC and presented on MHC class II for activation of CTL ○ Apoptosis is activated if the cell cycle is perturbed or by other stress such as damage to the membrane ○ Regulatory proteins (BCL-2 family) control this process which can proceed by either (i) permeabilisation of the mitochondria or (ii) direct signalling through TNF or FAS ○ Viruses require an intact and functional cell in which to replicate and apoptosis is a means of anti-viral defence Understand the strategies employed by viruses to avoid these responses Not being recognised by the immune system Interfering with functioning of particular immune mechanisms It is the large complex DNA viruses that have acquired many strategies for immune evasion since RNA viruses have limited coding capacity because of their small genome The viruses utilise components of the anti-viral mechanisms the host has to manipulate them and encourage a pro-viral condition Evasion of these responses can occur by ○ Completely disabling a pathway/process ○ Modulation of specific/process Evasion of the cellular immune response and its products ○ Antibody and T cells Latency (HSV in neurons, EBV in B cells) A non replicating cell is infected or the viral genome is replicated in conjunction with host DNA replication so the cell cycle is not disrupted 121 The viral genome persists intact so that the virus can be produced at a later time for transmission Expression of productive cycle viral genes is absent/inefficient → no structural protein, nothing expressed on the surface, only genome is present Immune detection of the cell with the latent genome is reduced or eliminated The herpesvirus grow productively in epithelial cells prior to becoming latent so evasion is in the context of an existing immune response - same for HIV in initial infection of T cells Other forms of viral persistence besides latency Chronic infections (hep B and C viruses) ○ HBV produces of decoy VLPs of surface antigen ○ HCV - quasispecies (many mutations and variation), immune exhaustion (see a lot of antigens and get overwhelmed) Non-cytopathic infections of inaccessible site (HPV) - delays or avoids robust immune response induction Acute with very late complications (measles - SSPE) ○ The immune response selects a variant of measles that under-expresses surface glycoproteins or matrix proteins and can no longer bud out from the cell to form free virions ○ The genome spreads gradually from neuron to neuron in the brain, possibly by the formation of an intercellular bridge, therefore escaping the immune response. ○ Antibody and/or T cells Antigenic variation in B or T cell epitopes Antigenic drift (influenza, HIV), and shift in influenza Change in the antigenic structure of the virus Due to selection of variant viruses with amino acid substitutions in the viral structural proteins that allow the virus to escape neutralisation by pre-existing antibody 122 Variant viruses arise spontaneously through errors in RNA replication giving rise to point mutations in the genes In influenza, antigenic drift occurs on a population scale with different mutations accumulating as the virus moves from person to person. In HIV infection a diversity of strains may develop within a single patient Drift in T cell epitopes Selective pressure of T cell immunity can lead to escape variants that avoid recognition due to mutations that: ○ Change the anchor residues of epitope so it no longer binds well to MHC ○ Change flanking amino acids so epitope is no longer processed from intact antigen ○ Change the epitope in TCR contact residues so it can be processed and presented but existing T cells can no longer recognise the presented peptide In chronic HCV patients, variants have been described with mutations in TCR contact residues for which there are no T cells with TCRs capable of interaction (exploitation of hole in the repertoire) ○ T cell priming by DC Blocking TLR signalling, DC maturation, interaction with naive T cells ○ TCR recognition 123 Antigenic variation in Tc epitopes (HIV, influenza) Inhibiting processing and presentation of viral peptides (many viruses) CMV protein US6 binds to TAP transporter on luminal side and prevents peptide translocation to ER HSV protein (ICP47) binds to cytosolic side of TAP transporter and prevents peptide translocation to ER HCMV protein US3 binds tapasin and inhibits optimal peptide loading Adenovirus protein E3 binds MHC peptide complex and retains it in ER and inhibits recruitment of TAP to the peptide loading complex EBNA-1 in latently infected B cells vades proteasome degradation Decreased production of MHC I molecules (HIV, RSV, adenovirus) HIV Nef protein induces endocytosis of class I molecules Class I gene transcription decreased - e.g HIV tat, RSV, adenovirus Norovirus - via translation repression intracellular MHC I is reduced ○ Less MHC I is transported to the surface, the infected cell is not recognised by CD8+ and not killed ○ NK cell recognition Mutations in ligand for activating receptor - MCMV Murine CMV encodes a protein (m152) which retains NKG2D ligands in the ER Virus encoded MHC class I like molecules - HCMV Human CMV encodes an MHC class I like molecule (UL18) which is expressed on surface of infected cells and delivers a negative signal to NK cell but cannot itself present peptides to CD8 T cells Upregulation of non-classical class I molecules - HCMV Human CMV upregulates the expression of the cellular non-classical HLA-E which is a class I molecule that only binds 124 and presents the signal peptides of other MHC molecules, NK cells see this as normal NK cell activity is controlled by a balance of stimulatory and inhibitory signals Viruses that cause reduction of MHC class I expression evade CD8+ T cell recognition but may become more susceptibleto NK killing ○ Interferon (type II IFN secreted by T cells) Flow on effects for T cell priming - IFNγ upregulates MHC class II in addition to class I ○ Other cytokines Virus encoded cytokine receptor homologues - pox viruses Poxviruses (vaccinia, myxoma) encode homologues of IFN-α/βR, IFNgR,TNF-αR and IL-1βR that bind the cytokine and inhibit its function In some cases the homologues can bind to monomeric receptors and inhibit formation of the active multimer Mutant viruses lacking these receptor homologues are greatly reduced in virulence Redirecting the T cell response - EBV EBV encodes an IL-10 homolog that suppresses a Th1 response (therefore reducing CTL) Intracellular blocking of cytokine production - IL-1β by crm A protein of vaccinia, cowpox Poxviruses encode crmA protein which inhibits the cleavage of IL-1 precursor to mature secreted form so virus-infected cell does not release IL-1 so inflammatory response is reduced Intracellular interference with cytokine function - TNFα and adenovirus Adenovirus encoded proteins interfere with the pathway of TNF- mediated killing of the cell. ○ Apoptosis modulation BCL-2 mimics, vFLIPS, caspase inhibitors and activators 125 ○ Autophagy modulation Some viruses encode proteins to block the execution of autophagy Other viruses utilise components of the autophagic machinery to foster their own replication or non-lytic cellular egress ○ Complement Virus encoded homologues of complement control proteins Complement pathways are normally carefully controlled by factors that positively and negatively regulate activation pathways These control proteins stop the C’ cascade by binding to C’ components Poxviruses and herpesviruses encode several proteins that are homologues of these control proteins and prevent C’ cascade e.g vaccinia protein binds C3b and C4b 126 Lecture 18 For a virus to persist it must maintain its genome within the infected host and avoid elimination by the immune response Latent infections - persistent infection with long periods without detectable virus and can reactivate, especially after immunosuppression ○ Viral recrudescence Why are persistent infections important ○ Epidemiological reasons - large and long lived pool of infected individuals. Promotes spread of low-infectivity viruses ○ Inapparent infections that may be reactivated ○ May lead to immunopathogenic diseases ○ Associated with neoplasms ○ May be established in vaccinated persons ○ Cannot be eradicated by antiviral chemotherapy Examples of different types of persistence ○ Acute with late complications (e.g. measles SSPE) ○ Latent infections (e.g. Herpesviruses) ○ Chronic infections (e.g. Papillomavirus, Hepatitis B, Hepatitis C) ○ Slow infections (e.g. HIV, prion diseases) 127 Herpesviruses found in virtually all species, all Herpesviruses form life-ling persistent infection (i.e latent) ○ Shows periodic reactivation - often (not always) following immunosuppresions and causes cytopathic infection ○ 150kb dsDNA genome, enveloped ○ Tegument layer where it packs all its viral proteins into an area between the membrane and capsid ○ Capsid contains the linear dsDNA genome - early, midearly, late, structural proteins ○ Tegument layer binds to receptor and releases the capsid and all the tegument proteins ○ VP16 translocates to nucleus with linear DNA ○ VP16 promotes early transcripts, the DNA circularises to form episomes assembles with histones ○ Immediate early protein turns on expression of early protein through early RNA ○ Early proteins drives replication of DNA 128 ○ Switch to express late transcription - accumulation of new DNA → anti-repression mechanism ○ Late proteins are structural proteins - capsid, envelope glycoproteins, tegument proteins ○ All these proteins assemble together with packaged up new DNA ○ Complex passage through ER-golgi network that places the glycoproteins on the surface of new virus particle that is then exocytosed Herpesvirus subfamilies ○ Alphaherpesviruses - Herpes simplex virus (HSV-1 and 2), Varicella-zoster virus (VZV - causes chickenpox and shingles) Neuronal persistence in humans ○ Betaherpesviruses - Cytomegalovirus (CMV) and Human herpesvirus 6 (HHV-6) monocyte/secretory gland persistence ○ Gammaherpesviruses - Epstein-Barr virus (EBV) and Kaposi sarcoma virus (HHV-8) Lymphocytes are site of persistence Herpesviral genetic properties ○ Shows regulated gene expression in lytic infection (diagram above) Early genes - include enzymes (HSV thymidine kinase) and regulatory genes (e.g. HSV viral host shut-off gene product), made before viral DNA replication Structural genes - components of viral capsid and envelope, made after viral DNA replication ○ Limited gene expression during latency LATs for HSV (latency associated transcripts) and EBNA-1-6 for EBV HSV-1 → 70-90% adults are seropositive, HSV-2 → 20-60%, CMV → 40-100%, EBV → 90% Viral genome persists as circular non-integrated DNA (episome) ○ Replication and non-replication latent genome ○ EBV - replicating latency Has two types of origins of replication 129 Ori-P (origin of plasmid maintenance) - used to replicate DNA during latent infection of dividing B cells → this ORI is binding site for latent gene product EBNA-1 Ori-Lyt - used to replicate virus DNA during lytic (productive) infection ○ HSV - non-replicating latency Uses OriL (long arm of genome) and OriS (short arm of genome) during lytic infection → exists on either end of the genome EBNA-1 initiates latent viral replication Primary surface HSV infection ○ Enters through a break in skin or mucosa ○ Local replication occurs where 85-90% of time inapparent infection (seropositive) 10-15% of time lesions form Less than 1% of time, spreads to blood and distant organs like the brain ○ In primary infection of HSV-1 causes gingivostomatitis in the mouth and involving lips and tongue 130 HSV - CNS diseases in humans ○ Very rare in adults ○ Most common during disseminated infection in the newborn - usually when mother has genital herpes virus and transfer of virus occurs ○ 70% fatal with 2.5% or less attaining normal neurological function ○ 50% of cases are primary infections by HSV-1, rest are recurrent HSV-1 infections ○ Now diagnosed by PCR on CSF and confirmed by viral culture HSV primary neuronal infection ○ Primary infection is important since replication occurs in the epithelial layer ○ Sensory neurons from the axon supplies innovation and sensory connection with those tissues, can take up via capsids with the dsDNA ○ Migration follows retrograde axonal transport to the sensory neuron cell body and in this site it is able to be maintained and replicate ○ Beyond the sensory neurons to the central nervous system does not seem to be a possible pathway for the virus particles ○ Innervations of blood vessels and sweat glands, sympathetic nervous system can also be a pathway ○ ○ HSV-1 recurs as herpes facialis (cold sore) on the mucocutaneous junction on the lip 131 HSV-1 - recurrent ○ 20% people harbour latent genome ○ Maintained episomally in neuronal cell bodies ○ Multiple genome copies per cell ○ No structural gene expression ○ Expression of 2kb latency activated mRNA transcripts (LATs) ○ No protein products identified from LATs ○ Anterograde axonal transport of virus - take the genomes from the cell body to the terminal end of the axon to appear as infectious viral particles LATs ○ Stable, non translated transcripts that are seen in latency and linked to reactivation ○ Not translated to protein ○ Processed into virus-derived miRNAs (viRNAs) 132 ○ Selection between replication and latency ○ Primary infection - retrograde transport Latency - no virus particles Reactivation - anterograde transport HSV-1 recurs orally due to latent replication in trigeminal ganglion HSV-2 recurs on the genitalia due to latent replication in the sacral ganglion ○ 40% of clinical isolates from genital sores are HSV-1, 5% of strains isolated from facial area are HSV-2 VZV’s primary infection causes the mild disease of chickenpox Primary infection phase, the virus is replicating at the skin and can enter the sensory neuron and travel to the dorsal root ganglion which is where it can maintain latency The virus can become reactivated and transfer back to the site where the virus first entered VZV can spread from one cell body to another through the dorsal root ganglion After long latency, a recurrent infection of VZV causes shingles that follow a dermatome 133 Zoster shingles lesions are frequently associated with immune deficit/decline → e.g. elderly Generally unilateral vesicles, commonly on the trunk or on the face following a ganglion’s dermatome A serious disseminated zoster may occur in the immunocompromised patients Therapeutic interventions ○ Antiviral chemotherapy - e.g. acyclovir ○ Passive immunisation with Zoster immunoglobulin Reactivation of VZV and shingles may occur in children and does not require immune deficit EBV causes infectious mononucleosis/glandular fever ○ Acquired by direct contact of infected saliva with oropharynx ○ Characterised by atypical lymphocytes - large pleomorphic blasts, basophilic vacuolated cytoplasm, lobulated nuclei, etc ○ Diagnosed by PCR EBV-1 predominates in Australia, USA and Europe EBV-2 and 1 equally prevalent in Africa Productive infection of epithelial cells of the nasopharynx and salivary glands ○ Circular (episomal DNA); a few proteins are continually expressed Infects B cells - no replication, site of latency Chronic primary EBV infection may cause B-lymphoproliferative diseases in transplant recipients and in vitro B cell culture 90% of humans are latent carriers of EBV 134 Primary site infection - oropharyngeal cavity → probably differentiated epithelial cells Initial infection is lytic and can spread to B cells where it generates widespread immune response and results in swelling of the lymph nodes ○ LMP2 is only expressed then the infected B cell is promoted for migration through the lamina propria back to infection sites where they can reactivate further into the lytic phase Latent EBV infection ○ Associated with B cell infection ○ Associated with limited expression of viral genes Epstein-Barr virus nuclear antigens (EBNA) 1-6 Latent membrane proteins (LMP) 1,2 ○ EBNA-1 is involved in replication of EBV genome in dividing B cells harbouring latent EBV infection EBV associated tumorigenesis ○ 90% humans have EBV LMP-2a mRNA+ memory B-cells ○ Reactivation of latent EBV from B-lymphocytes is associated with Hodgkin’s disease, Burkitt’s lymphoma and nasopharyngeal carcinoma 135 Burkitt’s B-cell lymphoma - common childhood cancer in Africa, cells express EBV EBNA-1 protein and have chromosome translocations involving c-myc and Ig Nasopharyngeal carcinoma - common in China, epithelial tumour cells express several EBV proteins: EBNA-1, LMP-1 and LMP-2 EBV associated with multiple sclerosis Cytomegalovirus ○ 40-80% of adult population seropositive ○ Infection in older children and adults leads to mild disease (fever/mononucleosis-type disease) ○ Results in persistent infection ○ Immunosuppression leads to reactivation of virus – seen in AIDS patients – major cause of death in bone-marrow transplant patients ○ Congenital infection can lead to deafness, chorioretinitis, and microcephaly, etc ○ Leading infectious cause of stillbirth 136 Lecture 19 Hepatitis is an inflammatory disease of the liver ○ Virus induced inflammation of the liver ○ Acute disease - weeks to months Non-specific, flu-like symptoms Jaundice, dark urine ○ Chronic disease - years to lifetime General malaise Cirrhosis, liver cancer ○ Fulminant disease Hepatitis viruses replicate in hepatocytes in the liver and the immune response to hepatitis viruses causes liver damage Hepatitis viruses are not cytolytic (don’t kill the infected cells), but are cytopathic Most disease is immune-mediated with age-related outcomes Exposure early in life results in less severe acute disease, higher rates of chronic infection One disease, many viruses ○ HAV - infectious hepatitis ○ HBV - serum hepatitis ○ HCV - non-A, non-B ○ HDV - dependent on HBV ○ HEV - enteric non-A, non-B ○ Different viral families to each other, no cross protection ○ HBV and HCV cause chronic infection 137 ○ HAV and HEV are acute viruses Diagnosis of acute viral hepatitis ○ Acute vs chronic or past infection ○ Serological tests - ELISAs, etc IgM antibody to viral proteins (acute) - reactive at 1-2 weeks, sensitivity more than 90%, specificity more than 99% IgG antibody to viral proteins - rising titre confirms acute infection ○ Nucleic acid tests - PCR from blood, faeces, but inferior to ELISA, does not give idea of whether this is acute/chronic/past infection Highest HAV notifications in Australia at NT HAV ○ Picornaviridaefamily - Hepatovirus ○ Non enveloped (+) ssRNA virus 30 nm particle resistant to stomach acid 7500 nt coding a single polyprotein ○ Single serotype worldwide ○ Infects man, many higher primates ○ Replicates in cell culture Non-cytolytic release of HAV virus particles ○ The HAV virion assembly is completed in multivesicular bodies and membrane encased viruses are released by exocytosis in endosomes containing up to four naked virion, but no membrane embedded viral protein 138 Life cycle of HAV and HEV ○ HAV clinical features ○ Incubation period - average o
